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Abstract

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Introduction

Pluripotent stem cells are the next-generation platform in various clinical and biological fields. They are a promising tool that can be used in disease modeling, drug screening, and regenerative medical therapy. Human Embryonic Stem Cells (hESCs) were mainly used to study and understand pluripotent cells. However, isolated by the destruction of the human blastocyst, hESCs are associated with several ethical concerns. In 2007, Dr. Shinya Yamanaka and his team reversed the cell programming process and developed stem cells from human adult somatic cells1,2. Therefore, unlike hESCs, induced-Pluripotent Stem Cells (iPSCs) can be generated from mature somatic cells, avoiding the ethical hurdles.

Usually, iPSCs are generated by the delivery of four exogenous genes: Oct4, Sox2, Klf4, and c-Myc. These Yamanaka factors are originally delivered using lentiviral and retroviral systems. The first iPSCs were derived from mouse somatic cells3. Afterwards, the technique was applied to human dermal fibroblasts1,2. Subsequent studies successfully generated iPSCs from various sources, such as urine4, blood5,6, keratinocytes7, and several other cell types. However, there are some somatic cells that have not been used in reprogramming, and screening of the reprogramming capabilities of various cell types from specific tissues in disease state, is still required.

Rheumatoid arthritis (RA) is a disease that can strike all joints and lead to autoimmune conditions in other organs. RA affects about 1% of adults in the developed world. It is a rather common disease and its incidence increases each year8. However, RA is hard to identify in the early stages and oncebone destruction occurs there is no treatment that can recover the damage. Moreover, drug efficacy differs from patient to patient, and it is hard to predict the medicine that is required. Therefore, the development of a drug-screening method is needed, and a cell material that can reflect the conditions of RA is required.

Fibroblast-like Synoviocytes (FLSs) are an active cellular participant in the pathogenesis of RA9,10. FLSs exist in the synovial intimal lining between the joint capsule and cavity, which is also referred to as the synovium. By supporting the joint structure and providing nutrients to the surrounding cartilage, FLSs usually play a crucial role in joint function and maintenance. However, FLSs in RA have an invasive phenotype. RA FLSs have a cancer-like phenotype, eventually destroying the surrounding bone by infinite proliferation10. With this unique characteristic, FLSs can be used as a promising material that can reflect the pathobiology of RA. Yet, these cells are rarely produced, and the cell phenotypes alter as the cells go through several passages in in vitro conditions. Therefore, it can be complicated to use RA FLSs as a tool that can represent the patient's condition.

Theoretically, RA patient-derived iPSCs (RA-iPSCs) can become an ideal tool for drug screening and further research. Generated iPSCs have self-renewal ability and can be maintained and expanded in vitro. With pluripotency, these cells can be differentiated into mature chondrocyte and osteocyte lineages, which can contribute cell material for specific research in RA and other bone-related diseases11.

In this study, we demonstrate how to isolate and expand FLSs from a surgically removed synovium, and how to generate RA-iPSCs from FLSs using lentiviruses containing Yamanaka factors.

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Protocol

Ethics Statement: This study protocol was approved by the institutional review board of The Catholic University of Korea (KC12TISI0861).

Replace the media with fresh media every 3 d. Split the cells at 80% confluency using 1 ml PBS/1 mM EDTA. Maintain until passage 3 before use. Divide each dish of cells into 3 dishes in every split.
​NOTE: After reaching passage 3, cells that are not going to be used immediately can be frozen.

2. Reprogramming FLSs Using Lentiviruses-encoding Yamanaka Factors

Transduction (D0)

Seed 3 × 104 cells per well of a 6-well plate in growth media (500 ml of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin). Incubate the cells O/N at 37 °C in 5% CO2.

The following day, remove one vial of lentivirus containing 4 Yamanaka factors: Oct4, Klf4, Sox2 and c-Myc from the freezer and thaw at 4 °C. Note: Lentivirus was produced by the procedure described in our previous study11.

The next day, replace the media with a mixture of FLS growth media and iPSC media (1:1 ratio) containing 0.1 mM sodium butyrate and 50 µg/ml ascorbic acid.
Note: The components of the iPSC media is given in the materials/equipment list.

Splitting Cells for Colony Formation

Prepare a vitronectin-coated 6-well plate.

Add 60 µl vitronectin to 6 ml PBS without Ca2+ and Mg2+. Put 2 ml of mixture into each wells and incubate in RT for at least 1 hr. Note: The working concentration of vitronectin is 5 µg/mL.

Split the cells at 3 different ratios (1:3, 1:6, and 1:9) to achieve different confluencies. Add 900 µl of media to the cell pellet and resuspend. Add 300, 150, and 100 µl of the cell mixture per well of a 6-well plate to achieve a ratio of 1:3, 1:6, and 1:9, respectively.

Replace the media daily with iPSC media until colonies appear. Colonies will appear after about D18. Note: At this stage, iPSC colonies co-exist with the non-reprogrammed FLSs.

Colony picking

Prepare a 48-well vitronectin-coated plate by adding 500 µl vitronectin to the wells, and incubate at RT for at least 1 hr.

Place the microscope on a clean bench, and remove the 6-well plate from the incubator.

Incubate the cells with the staining solution at RT for 15 min, avoiding light.

Discard the staining solution and rinse the cells with rinse buffer.

Cover the cells with PBS to prevent drying and verify expression using a bright-field microscope.

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Representative Results

In this study, we describe a protocol to generate iPSCs from FLSs using a lentiviral system. Figure 1A shows a simple scheme of the FLS isolation protocol. Following surgical removal of the synovium, the tissue was chopped into small pieces using surgical scissors. Collagenase was added to isolate the cells from the clumps of tissue. Cells were incubated for 14 days before further processing. Figure1B shows the morphology of the isolated FLSs. Cells were maintained for 3 passages before use. FLSs share a similar characteristic with general fibroblasts. Our isolated FLSs expressed the fibrotic markers, Vimentin and Fibronectin. The isolated RA FLSs also showed low expression of the macrophage-like synoviocyte marker, CD68 (Figure 1C).

FLSs were harvested and transduced with lentiviruses containing 4 Yamanaka factors for reprogramming. After splitting the cells at various ratios (D7), small colonies started to appear. Visible colonies, as shown in Figure 2A, appeared at D8-11. Colonies can be picked from this point. Figure 2B shows an image of a picked and amplified colony.

iPSCs were expanded until passage 5-10 and then used in various assays. Undifferentiated ESCs are characterized by a high level of AP. Cells were stained for AP to confirm the undifferentiated state. RA-iPSCs expressed AP (Figure 2C), indicating they are undifferentiated. Pluripotent marker expression was examined (Figure2D, E). The expression of pluripotent markers such as Oct3/4, Sox2, Nanog, Lin28, DPPB5 and TDGF1 was confirmed using RT-PCR (Figure 2D). The expression of Oct3/4, Sox2 was also confirmed by immunofluorescence analysis with additional markers such as SSEA4, TRA-1-60, TRA-1-81 and Klf4. TRA-1-60, which is currently thought to be the most important iPSC marker, was highly expressed in our generated iPSCs.

For further analysis, we performed karyotyping and teratoma assay. RA-iPSCs showed a normal chromosomal pattern of 44 + XY (Figure 3A). 12 weeks-post injection of RA-iPSCs into SCID mice, teratomas had formed and displayed diverse tissues, such as gland, adipose tissue and blood vessels (Figure 3B). The germ layer differentiation was also confirmed through immunofluorescence staining. Ectoderm lineage cells were positively stained for Otx2. Mesodermal cells expressed brachyury, and endoderm was confirmed by positive staining of SOX17.

Figure 1: Protocol for FLS Isolation from a RA Patient. (A) A simple diagram of the method used for synoviocyte isolation. (B) Morphology of isolated FLSs. (C) Fluorescence microscopy image of FLSs stained with fibrotic markers vimentin, fibronectin and a macrophage-like synoviocyte marker, CD68. All Scale bars = 200 µm. Please click here to view a larger version of this figure.

Figure 2:Generation of iPSCs from FLSs Isolated from a RA Patient. (A) Bright-field image of an iPSC colony before picking. (B) Image of a colony after picking. (C) Colony stained for AP. (D) PCR analysis of pluripotent markers. (E) Fluorescence microscopy image of iPSCs. The generated iPSCs expressed all the pluripotent markers. All Scale bars = 200 µm. Please click here to view a larger version of this figure.

Discussion

Before the discovery of iPSCs, scientists mainly used ESCs to study stem cell biology and other cell lineages through differentiation. However, ESCs originate from the inner mass of a blastocyst, which is an early-stage embryo. To isolate ESCs, destruction of the blastocyst is inevitable, raising ethical issues that are impossible to overcome. Moreover, although ESCs have stemness characteristics and pluripotency, they cannot be obtained from individuals and are sometimes not an ideal tool for personalized analysis and disease screening.

In 2007, Takahashi et al. generated iPSCs from human fibroblasts2. Theoretically, unlike ESCs, iPSCs can be generated from any adult somatic cells. With this advantage, iPSCs are thought to be the ideal tool for auto-cell transplantation. Also, with the concept of epigenetic memory, iPSCs are thought to be the ideal cell material for the simulation of pathogenic conditions, i.e. disease modeling. There have been many studies done using various types of cells such as blood cells, urine cells and more. Yet, there are many cell types, such as FLSs, in which reprogramming has not been conducted.

Arthritic diseases are major immune disorders that can cause permanent disability. RA is caused by chronic inflammation in the joints, eventually resultimg in bone and cartilage damage. It is hard to cure or reverse the damage especially because cartilage cannot regenerate in vivo. Therefore regenerative medicine using iPSCs are a new critical tool for the cure of RA. The bone and cartilage loss is also resulted in pannus formation. Pannus is a horn-like structure that can be seen in the histological image of the joint. The pannus is made by FLSs that proliferate limitlessly like cancer cells. Therefore, it is thought that the FLSs can reflect the pathological characteristics of the disease. In this study, we used patient FLSs to generate iPSCs.

FLSs are the major cell type that contribute to the pathogenesis of RA. As a cell that is mostly exposed to the inflammatory environment inside the synovial joint, our group thought that it can be used as a material that reflects the patient's disease condition. Therefore, using the earliest reprogramming method, we attempted to generate RA-specific iPSCs from FLSs, using the delivery of the 4 Yamanaka factors - Oct3/4, Sox2, Klf4 and c-Myc - by lentivirus. FLSs were isolated from the removed synovium. The trimming of synovium was critical when isolating FLSs. Bone and fat residues can make it more complicated to obtain pure FLSs. Also, the use of collagenase is inevitable for FLS isolation. It is important to use cells between passages 3-8. When FLSs reach passage 3, the Yamanaka factor containing lentiviruses are generated following the procedure mentioned in our earlier work11. Using the produced lentiviruses, we successfully generated RA FLS-derived iPSCs (RA-iPSCs). Pure iPSC clones were generated by the colony picking method. The RA-iPSCs expressed all the pluripotent transcription markers and had a normal karyotype. Furthermore, the RA-iPSCs were able to differentiate into all three germ layers according to the teratoma assay. Also, we have confirmed that the RA-iPSCs showed more mineralization when differentiated into osteogenic lineages in vitro (data not shown) 11.

However, there are some limitations to this method. Lentiviruses require genomic integration for reprogramming. Klf-4 and c-Myc are oncogenes and these two Yamanaka factors can facilitate tumor growth in vivo. Therefore, it may not be ideal to use these factors to generate materials for clinical applications. Furthermore, FLSs and skin fibroblasts are not difficult to obtain in clinics. As mentioned in various reports, skin fibroblasts (i.e. dermal fibroblasts) can only be obtained by punch biopsies. FLSs can be isolated only by an invasive surgical procedure and this surgery is performed on patients with severe hyperplasia who have undergone knee surgery. Therefore, there is no need to use FLSs when reprogramming a non-RA iPSC. Additionally, the process by which fibroblasts are prepared for reprogramming is difficult and time-consuming. FLSs share the same shortcomings as skin fibroblasts. Therefore, cell sources that are easier to handle and obtain are required.

For this reason, researchers have begun to search for an alternative cell source. A currently used material is blood cells. It is easy to draw blood and the isolation process is relatively simple and fast. Furthermore, there is a shift from the use of lentiviruses to tools that do not require genome-integration, such as Sendai viral systems, small molecules, and episomal plasmids.

Although FLSs cannot be used as a material for diverse individuals, it is still a great material when generating RA-iPSCs for research purposes. In the future, we are looking to generate a RA patient FLS-derived iPSC bank. RA patients individually show diverse reactions to drugs that are used for treatment. With several lines of RA iPSCs, we are hoping to generate a drug-screening cell-bank for RA patients. By screening all the drugs on each cell line, we may be able to predict which drug will work on which individual patient.

In conclusion, this protocol describes the application of iPSC technology to rheumatology. With this protocol, iPSCs can be generated from RA patient-derived FLSs, and the generated iPSCs have the required characteristics. These iPSCs can be used in clinical research, drug screening, disease modeling, and regenerative medicine for further investigations of the biology of RA.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

This work was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs (HI13D2188), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2013R1A1A1076125).